KR100795745B1 - - antibodies against insulin-like growth factor i receptor and uses thereof - Google Patents

Abstract

An antibody that binds to IGF-IR and inhibits the binding of IGF-I and IGF-II to IGF-IR, characterized by the following a), b), c) and d):

a) of IgG1 isotype,

b) the ratio of IC ₅₀ values of inhibition of binding of IGF-I to IGF-IR to inhibition of binding of IGF-II to IGF-IR is shown as 1: 3 to 3: 1,

c) In a cell phosphorylation assay using HT29 cells in medium containing 0.5% heat inactivated fetal bovine serum (FCS), compared to this assay performed without the antibody, IGF- at a concentration of 5 nM Inhibits IR phosphorylation by more than 80%,

d) in a cell phosphorylation assay using 3T3 cells that provide 400,000 to 600,000 molecules of IGF-IR per cell in medium containing 0.5% heat inactivated fetal bovine serum (FCS) compared to this assay performed without the antibody. If present, does not show IGF-IR stimulatory activity measured by IGF-IR phosphorylation at a concentration of 10 μM.

Description

Antibodies to Insulin-Like Growth Factor I Receptors and Their Uses {ANTIBODIES AGAINST INSULIN-LIKE GROWTH FACTOR I RECEPTOR AND USES THEREOF}

The present invention relates to antibodies against insulin-like growth factor I receptor (IGF-IR), methods for their preparation, pharmaceutical compositions containing said antibodies and their use.

Insulin-like growth factor I receptor (IGF-IR, EC 2.7.112, CD 221 antigen) belongs to the family of transmembrane protein tyrosine kinases (LeRoith, D., et al., Endocrin. Rev. 16 (1995) 143-163 And Adams, TE, et al., Cell Mol. Life Sci. 57 (2000) 1050-1063). IGF-IR binds with high affinity to IGF-I and initiates a physiological response to the ligand in vivo. IGF-IR also binds to IGF-II but has a slightly lower affinity. IGF-IR overexpression promotes neoplastic transformation of cells, and there is evidence that IGF-IR is involved in malignant transformation of cells and is therefore a useful target for the development of therapeutics for the treatment of cancer (Adams , TE, et al., Cell.Mol.Life Sci. 57 (2000) 1050-1063).

Antibodies to IGF-IR are well known in the art and are being studied for their antitumor effects in vitro and in vivo (Benini, S., et al., Clin. Cancer Res. 7 (2001) 1790-1797; Scotlandi, K., et al., Cancer Gene Ther. 9 (2002) 296-307; Scotlandi, K., et al., Int. J. Cancer 101 (2002) 11-16; Brunetti, A., et al., Biochem. Res. Commun. 165 (1989) 212-218; Prigent, SA, et al., J. Biol. Chem. 265 (1990) 9970-9977; Li, SL, et al., Cancer Immunol. Immunother. 49 (2000) 243-252 Pessino, A., et al., Biochem. Biophys. Res. Commun. 162 (1989) 1236-1243; Surinya, KH, et al., J. Biol. Chem. 277 (2002) 16718-16725; Soos, MA, et al., J. Biol. Chem., 267 (1992) 12955-12963; Soos, MA, et al., Proc. Natl. Acad. Sci. USA 86 (1989) 5217-5221; O'Brien, RM, et al., EMBO J. 6 (1987) 4003-4010; Taylor, R., et al., Biochem. J. 242 (1987) 123-129; Soos, MA, et al., Biochem. J. 235 (1986) 199-208; Li, SL, et al., Biochem.Biophys.Res.Commun. 196 (1993) 92-98; Delafontaine, P., et al., J. Mol.Cel Cardiol. 26 (1994) 1659-1673; Kull, FC Jr., et al. J. Biol. Chem. 258 (1983) 6561-6566; Morgan, DO, and Roth, RA, Biochemistry 25 (1986) 1364- 1371; Forsayeth, J. R., et al., Proc. Natl. Acad. Sci. USA 84 (1987) 3448-3451; Schaefer, E.M., et al., J. Biol. Chem. 265 (1990) 13248-13253; Gustafson, T.A., and Rutter, W.J., J. Biol. Chem. 265 (1990) 18663-18667; Hoyne, P.A., et al., FEBS Lett. 469 (2000) 57-60; Tulloch, P.A., et al., J. Struct. Biol. 125 (1999) 11-18; Rohlik, Q. T., et al., Biochem. Biophys. Res. Comm. 149 (1987) 276-281; And Kalebic, T., et al., Cancer Res. 54 (1994) 5531-5534; Adams, T. E., et al., Cell. Mol. Life Sci. 57 (2000) 1050-1063; Dricu, A., et al., Glycobiology 9 (1999) 571-579; Kanter-Lewensohn, L., et al., Melanoma Res. 8 (1998) 389-397; Li, S. L., et al., Cancer Immunol. Immunother. 49 (2000) 243-252). Antibodies to IGF-IR are also described, eg, in Arteaga, C.L., et al., Breast Cancer Res. Treatment 22 (1992) 101-106; And Hailey, J., et al., Mol. Cancer Ther. 1 (2002) 1349-1353, as described in many publications.

In particular, monoclonal antibodies against IGF-IR called αIR3 are widely used to study IGF-I mediated diseases such as IGF-IR mediated progression and cancer. Alpha-IR-3 is described by Kull, F.C., J. Biol. Chem. 258 (1983) 6561-6566. Meanwhile, about a hundred publications have been published covering the research and therapeutic uses of αIR3 on the antitumor effects of αIR3, alone and in combination with cell proliferation inhibitors such as doxorubicin and vincristine. have. αIR3 is a murine monoclonal antibody known to inhibit IGF-I binding to the IGF receptor but not to inhibit IGF-II binding to IGF-IR. αIR3 stimulates tumor cell proliferation and IGF-IR phosphorylation at high concentrations (Bergmann, U., et al., Cancer Res. 55 (1995) 2007-2011; Kato, H., et al., J. Biol. Chem. 268 (1993) ) 2655-2661). Other antibodies that inhibit IGF-II binding to IGF-IR more strongly than IGF-I binding (eg, 1H7, Li, SL, et al., Cancer Immunol. Immunother. 49 (2000) 243-252) Is present. An overview of the art for antibodies and their properties and characteristics is provided in Adams, T.E., et al., Cell. Mol. Life Sci. 57 (2000) 1050-1063.

Most of the antibodies described in the art are of mouse origin. Such antibodies, which are well known in the art, are not useful for the treatment of human patients without further modifications such as chimerization or humanization. Based on the above disadvantages, human antibodies are certainly preferred as therapeutic agents in the treatment of human patients. Human antibodies are well known in the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Pharmacol. 5 (2001) 368-374). Based on this description, human antibodies against a wide variety of targets can be prepared. Examples of human antibodies against IGF-IR are described in WO 02/053596.

However, there is still a need for antibodies to IGF-IR which have a convincing advantage for patients in need of anti-tumor therapy. A suitable advantage for the patient is simply the reduction of tumor growth caused by anti-tumor treatment and a marked prolongation of the progression time.

Summary of the invention

The present invention includes an antibody that binds to IGF-IR and inhibits the binding of IGF-I and IGF-II to IGF-IR, wherein the antibody is characterized by the following a), b), c) and d) do:

a) of IgG1 isotype,

b) the ratio of IC ₅₀ values of inhibition of binding of IGF-I to IGF-IR to inhibition of binding of IGF-II to IGF-IR is shown as 1: 3 to 3: 1,

c) In a cell phosphorylation assay using HT29 cells in medium containing 0.5% heat inactivated fetal bovine serum (FCS), IGF at a concentration of 5 nM when compared to this assay performed without the antibody. Inhibits IR phosphorylation by at least 80%, preferably at least 90%,

d) in a cell phosphorylation assay using 3T3 cells that provide 400,000 to 600,000 molecules of IGF-IR per cell in medium containing 0.5% heat inactivated fetal bovine serum (FCS) compared to this assay performed without the antibody. Case, it does not show IGF-IR stimulatory activity measured by PKB phosphorylation at a concentration of 10 μM.

Antibodies according to the invention show advantages for patients in need of anti-tumor treatment and provide for reduced tumor growth and marked prolongation of progression time. Antibodies according to the invention have novelty and inventive properties that benefit patients suffering from diseases associated with IGF deregulation, in particular tumor diseases. The antibodies according to the invention are characterized by the abovementioned properties. Therefore, the property specifically binds specifically to IGF-IR, inhibits the binding of IGF-I and IGF-II to IGF-IR at the rates mentioned above, is of IgG1 isotype, and even IGF-IR overexpressing cells Does not activate the IGF-IR signal even at a concentration 200 times its IC ₅₀ value. Antibodies that do not have "IGF-I mimetic activity" offer powerful advantages when used as therapeutic agents.

Preferably, the antibody according to the invention additionally induces at least 20% cell death of an IGF-IR expressing cell sample after 24 hours at a concentration of 100 nM of said antibody by ADCC.

Preferably, in addition, the antibodies according to the invention induce at least 20% cell death of an IGF-IR expressing cell sample after 4 hours at an antibody concentration of 100 nM by CDC.

Preferably, the antibody according to the invention at a concentration of 5 nM completely inhibits IGF-I mediated signal transduction of IGF-IR in tumor cells.

The invention also includes nucleic acids. The encoded polypeptide can be assembled with each other antibody chain as defined in a) and b) below:

a) an antibody heavy chain comprising CDR CDR1 (aa 31-35), CDR2 (aa 50-66) and CDR3 (aa 99-107) of SEQ ID NO: 1 or 3;

b) an antibody light chain comprising CDR CDR1 (aa 24-34), CDR2 (aa 50-56) and CDR3 (aa 89-98) of SEQ ID NO: 2 or 4.

The present antibodies are preferably monoclonal antibodies and, in addition, chimeric antibodies (human constant chains), humanized antibodies and particularly preferably human antibodies.

This antibody competes with antibody 18 to bind to IGF-IR human (EC 2.7.1.112, SwissProt P08069).

The antibody is further characterized by an affinity of 10 ⁻⁸ M (K _D ) or less, preferably from about 10 ⁻⁹ to 10 ⁻¹³ M.

The antibody preferably does not exhibit detectable concentration dependent inhibition of insulin that binds the insulin receptor.

Preferably, the present invention provides an antibody comprising a complementarity determining region (CDR) having the following sequences a) and b):

a) an antibody heavy chain comprising CDR CDR1 (aa 31-35), CDR2 (aa 50-66) and CDR3 (aa 99-107) of SEQ ID NO: 1 or 3;

b) an antibody light chain comprising CDR CDR1 (aa 24-34), CDR2 (aa 50-56) and CDR3 (aa 89-98) of SEQ ID NO: 2 or 4.

The antibody is preferably of the IgG1 type and therefore provides C1q complement binding and induces CDC. The antibody is further characterized by its ability to bind to IgGFc receptors and induce antibody dependent cellular cytotoxicity (ADCC).

Antibodies according to the invention significantly prolong the progression time and reduce tumor growth, as compared to vehicle treated animals in a suitable xenograft tumor model. The antibody inhibits the binding of IGF-I and IGF-II to IGF-IR in vitro and in vivo, preferably in an almost equivalent manner to IGF-I and IGF-II.

The present invention further provides hybridoma cell lines for preparing such antagonistic monoclonal antibodies according to the present invention.

Preferred hybridoma cell lines <IGF-1R> HUMAB Clone 18 (antibody 18) and <IGF-1R> HUMAB Clone 22 (antibody 22) according to the invention are described in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany Deposited.

Cell line Deposit number Deposit day <IGF-1R> HUMAB-Clone 18 DSM ACC 2587 10.04.2003 <IGF-1R> HUMAB-Clone 22 DSM ACC 2594 09.05.2003

Antibodies obtainable from such cell lines are preferred embodiments of the present invention.

The invention further provides nucleic acids encoding such antibodies, expression vectors containing said nucleic acids and host cells for recombinant production of such antibodies.

The present invention further provides methods for producing recombinants of such antibodies.

The present invention further provides a method of treating cancer comprising administering to a patient diagnosed with cancer (hence the need for anti-tumor treatment) an effective amount of an antagonist antibody against IGF-IR according to the present invention. The antibody may be administered alone, in a pharmaceutical composition, or alternatively in combination with a radiotoxic or cytotoxic treatment such as a cytotoxic agent or a prodrug thereof.

The invention further comprises the use of an antibody according to the invention for the treatment of cancer and for the preparation of a pharmaceutical composition according to the invention. In addition, the present invention includes a method for preparing a pharmaceutical composition according to the present invention.

The invention further encompasses pharmaceutical compositions containing a pharmaceutically effective amount of the antibody according to the invention, optionally together with a buffer and / or adjuvant useful for the formulation of the antibody for pharmaceutical purposes.

The present invention further provides pharmaceutical compositions comprising such antibodies in a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition may be included in an article of manufacture or kit.

The invention further comprises a vector containing a nucleic acid according to the invention, which is capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell.

The invention further comprises prokaryotic or eukaryotic host cells comprising the vector according to the invention.

The invention further comprises a method for producing a recombinant human antibody according to the invention, characterized by expressing the nucleic acid according to the invention in a prokaryotic or eukaryotic host cell and recovering the antibody from the cell. The present invention further encompasses antibodies obtainable by such recombinant methods.

The present invention further includes a method for screening one antibody against IGF-IR from a majority of antibodies to IGF-IR, wherein the method comprises IGF- per cell in medium containing 0.5% heat inactivated fetal bovine serum (FCS). Cell phosphorylation assays using 3T3 cells providing between 400,000 and 600,000 molecules of IR were performed with the antibody and compared with these assays without the antibody, IGF-IR measured by PKB phosphorylation at a concentration of 10 μM. Antibodies that do not exhibit stimulatory activity are selected. Preferably the antibody has one or more of the above mentioned additional properties.

The invention further includes a method of preparing a pharmaceutical composition, said method comprising cells using 3T3 cells that provide 400,000 to 600,000 molecules of IGF-IR per cell in medium containing 0.5% heat inactivated fetal calf serum (FCS). When phosphorylation assays were performed with the antibody and compared with such assays performed without the antibody, the antibody that did not exhibit IGF-IR stimulatory activity as measured by PKB phosphorylation at a concentration of 10 μM was selected and IGF-IR Selecting one antibody against IGF-IR from the majority of antibodies against, preparing the antibody by recombinant expression, recovering the antibody, and combining the antibody with a pharmaceutically acceptable buffer and / or adjuvant It is characterized by. Preferably the antibody has one or more of the above mentioned additional properties.

The term "antibody" includes, but is not limited to, various forms of antibody, including whole antibodies, antibody fragments, human antibodies, humanized antibodies, and genetic engineering antibodies, so long as they retain the properties according to the present invention.

An "antibody fragment" comprises a portion of a full length antibody, generally at least an antigen binding portion or variable region thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, immunotoxins, and multispecific antibodies formed from antibody fragments. In addition, the antibody fragments are characterized by the characteristics of the VH chain, ie with the VL chain or with the VL chain that binds to IGF-IR, ie with the VH chain in the functional antigen binding pocket, Therefore, it includes single chain polypeptides that provide the property of inhibiting IGF-I and IGF-II from binding to IGF-IR.

"Antibody fragments" also include such fragments which themselves cannot provide effector function (ADCC / CDC) but, in combination with appropriate antibody constant domain (s), then provide such function in a manner according to the present invention. .

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to the preparation of an antibody molecule of a single amino acid composition. Thus, the term “human monoclonal antibody” refers to an antibody that exhibits single binding specificity with variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is from a transgenic non-human animal, eg, a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene fused to immortal cells. It is prepared by hybridomas containing the obtained B cells.

The term “chimeric antibody” generally refers to a monoclonal antibody comprising at least a portion of a variable region produced by recombinant DNA technology, ie, binding regions from one source or species, and constant regions derived from different sources or species. To mention. Particularly preferred are chimeric antibodies comprising murine variable regions and human constant regions. Such murine / human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA fragments encoding murine immunoglobulin variable regions and DNA fragments encoding human immunoglobulin constant regions. Other forms of "chimeric antibodies" encompassed by the present invention are those that are classified or subclassified from those of the original antibody. Such "chimeric" antibodies are also referred to as "classified-converted antibodies". Chimeric antibody preparation methods now include conventional recombinant DNA and gene transfection techniques well known in the art. For example, Morrison, S.L., et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; See US Pat. Nos. 5,202,238 and 5,204,244.

The term “humanized antibody” refers to an antibody wherein the backbone or “complementarity determining region” (CDR) is modified to include CDRs of immunoglobulins of different specificity compared to that of the parental immunoglobulin. In a preferred embodiment, the murine CDRs were grafted into the skeletal region of a human antibody to prepare a "humanized antibody." See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; And Neuberger, M.S., et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those exhibiting sequences recognizing the antigens specified above for chimeric and bifunctional antibodies.

As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The variable heavy chain is preferably derived from germline sequence DP-50 (GenBank L06618) and the variable light chain is preferably derived from germline sequence L6 (GenBank X01668). The constant region of the antibody is a constant region of human IgG1 type. Such regions may be allotypic, for example Johnson, G., and Wu, T. T., Nucleic Acids Res. As long as the induction properties of ADCC and preferably CDC according to the invention are maintained, 28 (2000) 214-218, the databases referenced therein are useful.

As used herein, the term “recombinant human antibody” refers to a host cell or from a host cell, such as any human antibody produced, expressed, manufactured or isolated by recombinant means, such as SP2-0, NSO or CHO cells. It is intended to include antibodies isolated from an animal (eg, a mouse) transduced to a human immunoglobulin gene or antibody expressed using a recombinant expression vector transfected into it. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences in rearranged form. Recombinant human antibodies according to the invention are subject to somatic hypermutation in vivo. Therefore, the amino acid sequences of the VH and VL regions of recombinant antibodies are sequences derived from and related to human germline VH and VL sequences, while not naturally present in human antibody germline repertoire in vivo.

As used herein, “binding” means that the antibody has an affinity of about 10 ⁻¹³ to 10 ⁻⁸ M (K _D ) for IGF-IR, preferably about 10 ⁻¹³ to 10 ⁻⁹ M Mention of combining

As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

"Constant domains" do not directly relate to the binding of an antibody to an antigen, but to effector function (ADCC, complement binding, and CDC). The constant domains of the antibodies according to the invention are of the IgG1 type. Human constant domains having the above characteristics are described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), and Brueggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361; Love, T. W., et al., Methods Enzymol. 178 (1989) 515-527. Examples are shown in SEQ ID NOs: 5-8. Other useful and preferred constant domains are the constant domains of antibodies obtainable from hybridoma cell lines deposited with DSMZ for the present invention. Constant domains useful in the present invention provide complement binding. ADCC and optionally CDC are provided by a combination of variable and constant domains.

As used herein, “variable region” (variable region of light chain (VL), variable region of heavy chain (VH)) denotes each pair of light and heavy chains directly related to the binding of the antibody to the antigen. . The variable domains of the human light and heavy chains have the same general structure, each domain having four conserved regions whose sequences are widely conserved and linked by three “hypervariable regions” (or complementarity determining regions, CDRs). Include. The framework region takes the β-sheet arrangement and the CDRs may form a loop connecting the β-sheet structure. CDRs in each chain are maintained in their tertiary structure by framework regions and together with CDRs from other chains form antigen binding sites. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity / affinity of the antibodies according to the invention, thus providing a further object of the invention.

The term "hypervariable region" or "antigen-binding portion of an antibody", as used herein, refers to an amino acid residue of an antibody that is responsible for antigen-binding. Hypervariable regions include amino acid residues from the "complementarity determining regions" or "CDRs". “Framework” or “FR” regions are those of the variable domain region except for the hypervariable region residues as herein defined. Therefore, the light and heavy chains of the antibody comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, from the N- to the C-terminus. In particular, CDR3 of the heavy chain is a region that contributes mostly to antigen binding. CDR and FR regions are described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) and / or residues from the "hypervariable loop".

As used herein, the term “binds to IGF-IR” means that an in vitro assay, preferably an antibody binds to a surface, and the binding of IGF-IR is measured by Surface Plasmon Resonance (SPR). Means binding of the antibody to IGF-IR in a binding assay. Binding means a binding affinity (K _D ) of 10 ⁻⁸ M or less, preferably 10 ⁻¹³ to 10 ⁻⁹ M.

Binding to IGF-IR can be studied by the BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden). The affinity of binding is defined by the terms ka (rate constant for association of antibody from antibody / antigen complex), kd (dissociation constant), and K _D (kd / ka). Antibodies according to the invention exhibit a K _D of 10 ⁻¹⁰ M or less.

The binding of IGF-I and IGF-II to IGF-IR is also inhibited by the antibodies according to the invention. Inhibition is measured by IC ₅₀ in an assay for binding of IGF-I / IGF-II to IGF-IR on tumor cells. This assay is described in Example 7. In this assay, the amount of radioisotope labeled IGF-I or IGF-II or an IGF-IR binding fragment thereof bound to IGF-IR provided on the surface of the tumor cell (eg HT29) is antibody. Measure with or without increase in concentration. The IC ₅₀ value of the antibody according to the invention for the binding of IGF-I and IGF-II to IGF-IR is 2 nM or less and the IC ₅₀ value for binding of IGF-I / IGF-II to IGF-IR. The ratio of is about 1: 3 to 3: 1. IC ₅₀ values are measured as the mean or median of at least three independent measurements. A single IC ₅₀ value can be excluded from the category.

As used herein, the term “inhibit binding of IGF-I and IGF-II to IGF-IR” refers to IGF-IR presented on the surface of HT29 (ATCC HTB-38) tumor cells in an in vitro assay. It refers to inhibiting the binding of I ¹²⁵ -labeled IGF-I or IGF-II to. Inhibition means an IC ₅₀ value of 2 nM or less.

The term “IGF-IR expressing cell” refers to such cells that overexpress IGF-I receptors up to about at least 20,000 receptors / cell. Such cells are, for example, tumor cell lines such as NCI H322M or HT29, or cell lines that overexpress IGF-IR after transfection of expression vectors for IGF-IR (eg, 3T3 ATCC CRL1658). The amount of receptor per cell is measured according to Lammers, R., et al., EMBO J. 8 (1989) 1369-1375.

The term “inhibition of IGF-IR phosphorylation” refers to 3T3 cells which provide 400,000 to 600,000 molecules of IGF-IR per cell in medium containing 0.5% heat inactivated fetal bovine serum (FCS) when compared to an antibody-free cell phosphorylation assay. Refer to cellular phosphorylation assay using. Phosphorylation is detected by Western blotting using antibodies specific for tyrosine-phosphorylated proteins. This assay is described in Example 11. Thermal deactivation of the FCS is performed by short heating to 56 ° C. to deactivate the complement system.

The term “inhibition of PKB phosphorylation” uses 3T3 cells that provide 400,000 to 600,000 molecules of IGF-IR per cell in medium containing 0.5% heat inactivated fetal calf serum (FCS) when compared to an antibody-free cell phosphorylation assay. Reference is made to cell phosphorylation assays. Phosphorylation is detected by western blotting using antibodies specific for PKB phosphorylated at serine 473 of PKB (Akt 1, Swiss Prot Acc. No. P31749). This assay is described in Example 11.

The term “antibody-dependent cytotoxicity (ADCC)” refers to the lysis of human tumor target cells by an antibody according to the invention in the presence of effector cells. ADCC is measured in the presence of effector cells, such as freshly isolated PBMCs or effector cells, purified from smokescreens, such as monocytes or NK cells, preferably by treating the preparation of IGF-IR expressing cells with an antibody according to the invention. ADCC is found when the antibody induces lysis (cell death) of at least 20% of tumor cells after 24 hours at a concentration of 100 nM. If ADCC is found more prominently at 4 h than 24 h, the measurement is performed at 4 h. The assay is preferably performed by measuring ⁵¹ Cr or Eu labeled tumor cells, specifically released ⁵¹ Cr or Eu. Controls include incubating tumor target cells with effector cells, but without antibodies.

The term "complement-dependent cytotoxicity (CDC)" refers to lysis of human tumor target cells by an antibody according to the invention in the presence of complement. CDC is preferably determined by treating a sample of IGF-IR expressing cells with the antibody according to the invention in the presence of complement. CDC is found when the antibody induces lysis (cell death) of at least 20% of tumor cells after 4 hours at a concentration of 100 nM. The assay is preferably performed by measuring ⁵¹ Cr or Eu labeled tumor cells, specifically released ⁵¹ Cr or Eu. Controls include incubating tumor target cells with complement but without antibodies.

The term “complete inhibition of IGF-I mediated signal transduction” refers to the inhibition of phosphorylation of IGF-I-mediated IGF-IR. For this assay, IGF-IR expressing cells, preferably H322M cells, are stimulated with IGF-I and treated with the antibodies according to the invention (antibody concentrations of 5 nM or higher are useful). SDS PAGE is then performed, and Western blotting analysis with antibodies specific for phosphorylated tyrosine to determine phosphorylation of IGF-IR. If no band, referred to as phosphorylated IGF-IR, on the western blot is detected visually, complete inhibition of signal transduction is found.

Antibodies according to the invention exhibit binding to the same epitope of IGF-IR as antibody 18 or inhibit binding to IGF-IR due to steric hindrance of binding by antibody 18. Binding inhibition can be detected by SPR assays using immobilized antibody 18 at concentrations of 20-50 nM and IGF-IR and antibodies detected at concentrations of 100 nM. A signal reduction of at least 50% indicates that the antibody competes with antibody 18. Such assays can be performed in the same manner using antibody 22 as an immobilized antibody.

The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of chemically active surface groups of molecules such as amino acids or sugar side chains and typically have specific three dimensional structural characteristics, as well as specific charge characteristics. Arranged and unarranged epitopes are distinguished by the loss of binding to the former, with the exception of the latter in the presence of a denatured solvent.

Antibodies according to the invention furthermore include such antibodies with "conservative sequence modifications", and nucleotide and amino acid sequence modifications which do not affect or alter the above mentioned features of the antibodies according to the invention. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include replacing amino acid residues with amino acid residues having similar side chains. Families of amino acid residues having similar side chains are defined in the art. The family includes basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine , Tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. For example, tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues selected in human anti-IGF-IR antibodies may preferably be substituted with another amino acid residue from the same side chain family.

Amino acid substitutions are described in Riechmann, L., et al., Nature 332 (1988) 323-327 and Queen, C., et al., Proc. Natl. Acad. Sci. By mutagenesis based on molecular modeling as described by USA 86 (1989) 10029-10033.

In a preferred embodiment of the invention, the antibody according to the invention binds to the same epitope to which the binding parameters ka, kd and K _D , antibodies 18 and 22 bind, IGF-I and IGF- to IGF-IR on tumor cells IC ₅₀ values for inhibition of binding of II, and characterized in the tumor cells in addition by one or more features selected from the group selected from the IC ₅₀ values for the inhibition of the IGF-IR phosphorylation by stimulation of IGF-I It is erased. Inhibition of phosphorylation of IGF-IR results in inhibition of phosphorylation of downstream elements such as PKB, down-regulation of IGF-IR in tumor cells, and the three-dimensional growth phase of tumor cells in vitro. Antibodies are preferably further characterized by their pharmacokinetic and pharmacodynamic values, and cross-reactivity to other species.

The antibodies according to the invention inhibit the phosphorylation of tyrosine of IGF-IR and preferably also the phosphorylation of tyrosine of PKB to a similar extent.

Antibodies according to the invention preferably downregulate IGF-IR protein concentrations in tumor cells and tumors, eg, xenograft tumors.

The antibody according to the invention preferably inhibits the proliferation of IGF-IR expressing cells (eg NIH 3T3 cells) as well as three-dimensional growth of tumor cells in colony forming assays.

The antibody according to the invention does not inhibit the binding of insulin to the insulin receptor, preferably in a binding competition assay on insulin receptor overexpressing 3T3 cells using the antibody at a concentration of 200 nmol / l.

The antibody according to the invention is preferably produced by recombinant means. Such methods are well known in the art, and include protein isolation in prokaryotic and eukaryotic cells, with subsequent isolation of antibody polypeptides and purification, usually in pharmaceutically acceptable purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into the expression vector by standard methods. Expression is performed in suitable prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, yeast or E. coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). do.

Recombinant preparation of antibodies is well known in the art and described, for example, in Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R. J., Mol. Biotechnol. 16 (2000) 151-161; Werner, R. G., Drug Res. 48 (1998) 870-880.

Antibodies can be present in whole cells, cell lysates, or in partially purified or substantially pure form. Alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other well known in the art to remove other cellular components or other contaminants, such as other cellular nucleic acids or proteins. Purification is carried out by standard techniques, including conventional techniques. Ausubel, F., et al., Ed. See Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Expression in NSO cells is described, eg, in Barnes, L.M., et al., Cytotechnology 32 (2000) 109-123; And Barnes, L.M., et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described, for example, in Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of various domains is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; And Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. Preferred transient expression systems (HEK 293) include Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.

Suitable control sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.

A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a preprotein involved in secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site, when positioned to facilitate translation, is operably linked to a coding sequence. In general, “operably linked” means that the DNA sequences to which they are linked are contacted, and in the case of secretion diagrams, in the translation frame. However, enhancers should not be touched. Ligation is performed by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

Monoclonal antibodies are suitably isolated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. DNA and RNA encoding monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can be used as a source of such DNA and RNA. Once isolated, the DNA can be inserted into an expression vector, which is then transfected into a host cell, such as HEK 293 cells, CHO cells, or myeloma cells, which otherwise does not produce immunoglobulin proteins, resulting in recombination in the host cell. Synthesis of monoclonal antibodies is obtained.

Amino acid sequence variants of human IGF-IR antibodies are prepared by introducing suitable nucleotide alterations into the antibody DNA, or by peptide synthesis. However, this modification can only be carried out in a very limited range, for example as described above. For example, modifications do not alter the above-described antibody characteristics, such as IgG isotypes and epitope binding, but can facilitate recombinant preparation, improve yield of protein stability, or facilitate purification.

Any cysteine residue that is not involved in maintaining the proper alignment of the anti-IGF-IR antibody may also be generally substituted with serine to enhance the oxidative stability of the molecule and to suppress abnormal crosslinking. In contrast, cysteine binding can be added to the antibody to improve its stability (especially when the antibody is an antibody fragment such as an Fv fragment).

Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically N-linked. N-linking refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to the side chains of asparagine residues. Therefore, the presence of one of the tripeptide sequences in the polypeptide creates a potential glycosylation site. Adding a glycosylation site to the present antibody is conveniently carried out by altering the amino acid sequence to contain one or more of the above described tripeptide sequences (for N-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of anti-IGF-IR antibodies are prepared by a variety of methods known in the art. The method can be used to isolate or oligonucleotide-mediated (or specific site) mutagenesis from natural sources (for naturally occurring amino acid sequence variations), PCR mutagenesis, and previously prepared variants of humanized anti-IGF-IR antibodies. Or preparation by cassette mutagenesis of a non-variant version.

The present invention also provides prodrugs of chemotherapeutic agents, toxins (eg, enzyme-activated toxins of bacterial, fungal, plant or animal origin, or fragments thereof), radioisotopes (ie, radioconjugates) or cytotoxic agents. Suitable for immunoconjugates comprising an antibody according to the invention conjugated with a cytotoxic agent such as Agents useful for the generation of such immunoconjugates are described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, abrin A chain, mode Modeccin A chain, alpha-sarcin, varnish (Aleuritesfordii) protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordica Karan Momordica charantia inhibitors, curtin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin , Phenomycin, phenomycin, enomycin, and tricotecene.

Conjugates of antibodies and cytotoxic agents include N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters; (E.g., dimethyl adimimidate HCL), active esters (e.g. disuccinimidyl suverate), aldehydes (e.g. glutaaldehyde), bis-azido compounds (e.g. bis (p-azidobenzoyl) hexanediamine ), Bis-diazonium derivatives (eg, bis- (p-diazoniumbenzoyl) -ethylenediatin), diisocyanates (eg tolyene 2,6-diisocyanate), and bis-active fluorine compounds (eg 1 (5-difluoro-2,4-dinitrobenzene), such as various difunctional protein coupling agents. For example, lysine immunotoxins can be prepared as described in Vitetta, E.S., et al., Science 238 (1987) 1098-1104. Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotides to antibodies. See WO 94/11026.

Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. This procedure is beneficial because it does not require the preparation of an antibody in a host cell having glycosylation capacity for N- or O-linked glycosylation. Depending on the coupling scheme used, the sugar may be selected from (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) serine, threonine or hydroxyproline Free hydroxyl groups, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) amide groups of glutamine. The method is described in WO 87/05330, and Aplin, J.D., and Wriston, J.C. Jr., CRC Crit. Rev. Biochem. (1981) 259-306.

Removal of any carbohydrate moiety present on the antibody can be performed chemically or enzymatically. Chemical deglycosylation requires exposing the antibody to the compound trifluoromethanesulfonic acid, or equivalent compound. The treatment cleaves most or all sugars except the linking sugars (N-acetylglucosamine or N-acetylgalactosamine) without damaging the antibody. Chemical deglycosylation is described by Sojahr, H.T., and Bahl, O.P., Arch. Biochem. Biophys. 259 (1987) 52-57 and Edge, A.S., et al. Anal. Biochem 118 (1981) 131-137. Enzymatic cleavage of the carbohydrate moiety on the antibody is described by Thotakura, N.R., and Bahl, O.P., Meth. Enzymol. 138 (1987) 350-359, as described by the use of a variety of inner- and outer-glycosidase.

Another type of covalent modification of antibodies is described in US Pat. No. 4,640,835; No. 4,496,689; No. 4,301,144; No. 4,670,417; Linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner mentioned in US Pat. No. 4,791,192 or 4,179,337.

In another aspect, the present invention provides B-cells isolated from transgenic non-human animals, eg, transgenic mice expressing human anti IGF-IR antibodies according to the present invention. Preferably, isolated B cells are obtained from transgenic non-human animals, eg, purified or enriched preparations of IGF-IR antigens and / or transgenic mice immunized with cells expressing IGF-IR. Preferably, the transgenic non-human animal, such as a transgenic mouse, has a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or some antibodies of the invention. Isolated B-cells are then immortalized to provide a source of human anti-IGF-IR antibody (eg, hybridomas). Accordingly, the present invention also provides hybridomas from which the human monoclonal antibodies according to the present invention can be prepared. In one embodiment, the hybridoma is a transgenic non-human animal fused to an immortal cell, eg, a genome comprising a human heavy chain transformation gene and a human light chain transformation gene encoding all or some antibodies of the invention. B cells obtained from a transgenic mouse having a.

In one specific embodiment, the transgenic non-human animal is a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or some antibodies of the invention. Transgenic non-human animals may be immunized with purified or concentrated preparations of IGF-IR antigens and / or cells expressing IGF-IR. Preferably, transgenic non-human animals, such as transgenic mice, can prepare IgG1 isotypes of human monoclonal antibodies against IGF-IR.

Human monoclonal antibodies according to the invention are transgenic non-human animals, for example transgenic mice having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or some antibodies of the invention. Can be prepared by immunization with purified or concentrated preparations of IGF-IR antigen and / or cells expressing IGF-IR. Animal B cells (eg, splenic B cells) are then obtained and fused with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies against IGF-IR.

In a preferred embodiment, human monoclonal antibodies against IGF-IR can be generated using transgenic mice having parts of the human immune system rather than the mouse immune system. Such transgenic mice, referred to herein as “HuMAb” mice, include heavy chains (μ and γ) and κ light chains (constant region genes), with targeted mutations that inactivate endogenous μ and κ chain loci. It contains a human immunoglobulin gene microgenic locus encoding an unrearranged human immunoglobulin gene (Lonberg, N., et al., Nature 368 (1994) 856-859). Thus, mice exhibit reduced expression of mouse IgM or K, and in response to immunization, introduced human heavy and light chain transduction undergoes class conversion and somatic mutations, resulting in high affinity human IgG monoclonal antibodies (Lonberg , N., et al., Nature 368 (1994) 856-859; reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113 (1994) 49-101; Lonberg, N., and Huszar, D., Intern. Rev. Immunol 25 (1995) 65-93 and Harding, F., and Lonberg, N., Ann.N. Acad. Sci 764 (1995) 536-546). The manufacture of HuMAb mice is described in Taylor, L., et al., Nucleic Acids Research 20 (1992) 6287-6295, the entire contents of which are incorporated herein by reference; Chen, J., et al., International Immunology 5 (1993) 647-656; Tuaillon, N., et al., Proc. Natl. Acad. Sci USA 90 (1993) 3720-3724; Choi, T. K., et al., Nature Genetics 4 (1993) 117-123; Chen, J., et al., EMBO J. 12 (1993) 821-830; Tuaillon, N., et al., Immunol. 152 (1994) 2912-2920; Lonberg, N., et al., Nature 368 (1994) 856-859; Lonberg, N., Handbook of Experimental Pharmacology 113 (1994) 49-101; Taylor, L., et al., Int. Immunol. 6 (1994) 579-591; Lonberg, N., and Huszar, D., Intern. Rev. Immunol. 25 (1995) 65-93; Harding, F., and Lonberg, N., Ann. N. Acad. Sci 764 (1995) 536-546; Fishwild, D.M., et al., Nat. Biotechnol. 14 (1996) 845-851. Further, US Pat. No. 5,545,806; 5,569,825; 5,569,825; 5,625,126; 5,625,126; 5,633,425; 5,633,425; 5,789,650; 5,789,650; No. 5,877,397; No. 5,661,016; 5,814,318; 5,814,318; 5,874,299; 5,874,299; 5,545,807; No. 5,770,429; WO 98/24884; WO 94/25585; WO 93/1227; WO 92/22645; And WO 92/03918.

To generate fully human monoclonal antibodies against IGF-IR, HuMAb mice were cultured in Lonberg, N., et al., Nature 368 (1994) 856-859; Fishwild, D.M., et al., Nat. Biotechnol. 14 (1996) 845-851 and according to the general method as described by WO 98/24884 can be immunized with purified or concentrated preparations of IGF-IR antigen and / or cells expressing IGF-IR. Preferably, mice will be 6-16 weeks of age in primary immunity. For example, purified or concentrated preparations of soluble IGF-IR antigen (eg, purified from IGF-IR-expressing cells) can be used to immunize intraperitoneally in HuMAb mice. If immunization using purified or concentrated preparations of IGF-IR antigens does not result in antibodies, mice can also be immunized with cells expressing IGF-IR, eg, tumor cell lines, to promote an immune response. Experiments accumulated with various antigens were first immunized intraperitoneally (i.p.) with the antigen in the Freund's fully immune response adjuvant followed by biweekly with the antigen in the Freund's incomplete immune response adjuvant. When immunized (eg total 6 weeks or less), HuMAb transgenic mice show the best response. The immune response can be monitored with plasma samples obtained by the eye and blood after the course of the immune protocol. Plasma can be screened by ELISA and mice with sufficient titer of anti-IGF-IR human immunoglobulin can be used for immortalization of corresponding B cells. Mice can be intravenously enhanced with antigen 3 to 4 days prior to sacrifice and removal of the spleen and lymph nodes. It is expected that 2-3 fusions for each antigen will be needed to perform. Multiple mice will be immunized for each antigen. For example, a total of 12 HuMAb mice of the HCo7 and HCo12 strains can be immunized.

HCo7 mice have JKD disruption in their endogenous light chain (kappa) gene (as described in Chen, J., et al., EMBO J. 12 (1993) 821-830), CMD disruption in their endogenous heavy chain gene (WO 01 / 14424), KCo5 human kappa light chain transformation gene (as described in Fishwild, DM, et al., Nat. Biotechnol. 14 (1996) 845-851), and HCo7 human heavy chain transformation Gene (as described in US Pat. No. 5,770,429).

HCo12 mice have JKD disruption in endogenous light chain (kappa) genes (as described in Chen, J., et al., EMBO J. 12 (1993) 821-830), CMD disruption in their endogenous heavy chain genes (WO 01/14424). ), KCo5 human kappa light chain transformation gene (as described in Fishwild, DM, et al., Nat. Biotechnol. 14 (1996) 845-851), and HCo12 human heavy chain transformation gene (As described in Example 2 of WO 01/14424).

Mouse lymphocytes can be isolated using PEG based on standard protocols and fused with mouse myeloma cell lines to generate hybridomas. The resulting hybridomas are then screened for the preparation of antigen-specific antibodies. For example, single cell suspensions of spleen and lymph node-induced lymphocytes from immunized mice are fused with 50% PEG to 1/6 numbers of SP 2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581). Cells are plated at approximately 2 × 10 ⁵ in a flat bottom microtiter plate and then incubated for about two weeks in selection medium.

Individual wells are then screened by ELISA against human anti-IGF-IR monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, usually 10-14 days later, the medium is analyzed. Anti-IGF-IR monoclonal antibodies can be subcloned at least twice by limiting dilution if the antibody secreting hybridomas are replated, screened again, and still positive for human IgG. Stable subclones are then cultured in vitro to prepare antibodies in tissue culture medium for characterization.

Since the CDR sequences are responsible for antibody-antigen interactions, it is possible to express recombinant antibodies according to the invention by constructing an expression vector comprising the CDR sequences according to the invention on framework sequences from different human antibodies (eg See, for example, Riechmann, L., et al., Nature 332 (1998) 323-327; Jones, P., et al., Nature 321 (1986) 522-525; and Queen, C., et al., Proc. Natl. Acad. USA 86 (1989) 10029-10033. Such framework sequences can be obtained from public DNA databases comprising germline human antibody gene sequences. Since the germline sequences will not contain fully assembled variable genes, they will be different from mature antibody gene sequences, which are formed by V (D) J joining during B cell maturation. The germline gene sequence will also differ from the sequence of the high affinity secondary repertoire antibody, evenly across each variable region.

The present invention preferably comprises a nucleic acid fragment encoding a polypeptide that binds to IGF-IR, wherein the polypeptide is selected from the group consisting of a) and b), IGF-I and IGF-I for IGF-IR. Inhibit the binding of II:

a) an antibody heavy chain comprising CDR CDR1 (aa 31-35), CDR2 (aa 50-66) and CDR3 (aa 99-107) of SEQ ID NO: 1 or 3;

b) an antibody light chain comprising CDR CDR1 (aa 24-34), CDR2 (aa 50-56) and CDR3 (aa 89-98) of SEQ ID NO: 2 or 4.

The reconstructed heavy and light chain variable regions are combined with the sequences of promoter, translation initiation, constant region, 3 ′ untranslated, polyadenylation, and transcription termination to form expression vector constructs. The heavy and light chain expression constructs are combined into a single vector and then co-transfected, a series of transfections, or individual transfections into fused host cells to form a single host cell expressing both chains. do.

Thus, the present invention provides a method for producing a recombinant human antibody according to the present invention, characterized by expressing a nucleic acid encoding the following a) and b):

a) an antibody heavy chain comprising CDR CDR1 (aa 31-35), CDR2 (aa 50-66) and CDR3 (aa 99-107) of SEQ ID NO: 1 or 3;

b) an antibody light chain comprising CDR CDR1 (aa 24-34), CDR2 (aa 50-56) and CDR3 (aa 89-98) of SEQ ID NO: 2 or 4.

The invention further comprises the use of the antibody according to the invention for the diagnosis of in vitro IGF-IR, preferably by immunological assays measuring the binding between the IGF-IR of the sample and the antibody according to the invention. do.

In another aspect, the present invention provides a pharmaceutical composition comprising one or a combination of human monoclonal antibodies of the invention, or antigen-binding portion thereof, formulated with a composition, eg, a pharmaceutically acceptable carrier. To provide.

The pharmaceutical compositions of the invention may also be administered in combination therapy, ie in combination with other agents. For example, combination therapies can include the compositions of the invention in combination with one or more anti-tumor agents or other conventional therapies.

A "chemotherapeutic agent" is a chemical compound useful for treating cancer. Examples of chemotherapeutic agents include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa ), Taxotere (docetaxel), Busulfan, Gemcitatabine, Cytoxin, Taxol, Methotrexate, Cisplatin, Mel Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincrastin Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin ), Mitomycins, esperamicins (US Pat. No. 4,675,187), melphalan and other related nitrogen mustard.

As used herein, the term “cytotoxic agent” refers to a component that inhibits or prevents the function of a cell and / or causes cell destruction. The term is intended to include toxins such as radioisotopes, chemotherapeutic agents, and bacterial fungi, enzymatically active toxins of plant or animal origin, or fragments thereof.

As used herein, the term "prodrug" refers to a precursor of a pharmaceutically active ingredient, which is less cytotoxic to tumor cells than the parent drug and can be enzymatically activated or converted into a more active parental form or Reference is made to the derivative form. For example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pages 375-382, 615th edition Meeting Belfast (1986), and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug See Delivery, Borchardt et al., (Ed.), Pages 247-267, Humana Press (1985). Prodrugs of the present invention are phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acids, which can be converted to more active, non-cytotoxic drugs. Modified prodrugs, glycosylated prodrugs, (β-lactam ring prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and others Examples of cytotoxic drugs that may be derivatized in the form of prodrugs for use in the present invention include, but are not limited to, 5-fluorouridine prodrugs. Do not.

As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epithelial administration (eg by injection or infusion).

"Pharmaceutically acceptable salts" refer to salts that retain the desired biological activity of the antibody and do not confer any undesirable toxin effect (eg, Berge, SM, et al., J. Pharm. Sci. 66 (1977) 1-19). Such salts are included in the present invention. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochlorides.

The compositions of the present invention can be administered by a variety of methods known in the art. As assessed by those skilled in the art, the route and / or mode of administration will vary depending upon the desired result.

In order to administer a compound of the present invention by a particular route of administration, it will be necessary to coat the compound with a substance to prevent its deactivation, or to co-administer with the compound. For example, the compound may be administered to the patient in a suitable carrier, such as liposomes, or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffers.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the instant preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active ingredients is known in the art.

As used herein, the phrases "parenteral administration" and "administered parenterally" refer to the mode of administration, usually by injection, and not by intestinal and topical administration, without limitation, intravenous, intramuscular, intraarterial, intradermal , Intraarticular, orbital, intracardia, intradermal, intraperitoneal, bronchial, subcutaneous, subcutaneous, intraarticular, subarticular, subretinal, subspinal, intradural and intrasternal injections and infusions.

The composition may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms can be assured both by the sterilization process and by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, delayed absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

Regardless of the route of administration chosen, the compounds of the invention and / or the pharmaceutical compositions of the invention, which can be used in a suitable hydrated form, are formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage concentration of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective to achieve a desired therapeutic response for a particular patient, composition, and mode of administration, without toxicity to the patient. . The dosage concentration selected is the activity of the specific composition of the invention used, the route of administration, the time of administration, the rate of release of the specific compound used, the duration of treatment, the other drug, compound and / or substance used in combination with the particular composition used And various pharmacokinetic factors, including factors well known in the medical arts, including age, sex, weight, condition, general health and previous medical history of the patient being treated.

The composition must be sterile and fluid to the extent that the composition can be delivered by syringe. In addition to water, the carrier is preferably an isotonic saline buffer.

Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases, it is desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

The following examples, references, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the processes mentioned without departing from the spirit of the invention.

Brief description of sequence listing

SEQ ID NOs: 1 to 4 nucleotide and amino sequences of the light and heavy chain variable region domains of antibodies 18 and 22

SEQ ID NOs: 5 and 6 nucleotide and amino sequences of the human constant region domains

1 IGF-IR surface expression in low density and high density cell cultures.

2 WST assay (NCI H322M cells) for proliferation inhibition in 3D culture.

3 Colony formation assay (micrograph) for inhibition of proliferation in 3D culture.

4 Colony formation assay (quantification) for inhibition of proliferation in 3D culture. ctr = control; Cl 18 = Antibody 18.

Inhibition of I ¹²⁵ -IGF-I binding to HT29 cells by antibodies 18 and 22 (IC ₅₀ value).

Inhibition of I ¹²⁵ -IGF-II binding to HT29 cells by antibody 18.

Inhibition of I ¹²⁵ -IGF-II binding to HT29 cells by antibody αIR3.

8 Blocking phosphorylation of both IGF-I induced IGF-IR and Akt / PkB.

9 I ¹²⁵ -insulin binding to 3T3-IR cells is not inhibited by anti-hIGF-1R antibody. (MAX w / o Ab: maximum binding of I ¹²⁵ -insulin; MIN: minimum binding after competition with 1 μM insulin)

10 Does not stimulate the activity of AK18 on IGF-IR overexpressing 3T3 cells.

11 does not stimulate the activity of AK18 on human tumor cells (HT29).

12 Downregulation of IGF-IR exposed on H322N cell surface by antibody 18 in vitro.

Figure 13 Treatment with AK18, Stage 1 Tumor Volume

14 Treatment with AK18 with or without gemcitabine

Figure 15 Treatment with AK18, Stage 1 Tumor Volume

Example  One

term- IGF - IR  To manufacture antibodies Hybridoma  Cell line generation

Hybridoma  culture

The resulting HuMAb hybridomas were incubated at 37 ° C., 5% CO ₂ in Hybridoma Express Medium (PAA Laboratories GmbH, Austria) supplemented with 2 mM L-glutamine (BioWhittaker) and 4% Origen Cloning Factor (Igen, France). It was.

Immune Process in Transgenic Mice

1 x 10 ⁶ NIH 3T3 cells transfected with 10 HCo7 transgenic mice (4 males and 6 females), line GG2201 (Medarex, San Jose, CA, USA) with an expression vector for IGF-IR And alternate immunization with 20 μg of soluble extracellular domain of IGF-IR. Three intraperitoneal (IP) immunizations with IGF-IR expressing cells and three subcutaneous (SC) immunizations at the bottom of the tail with six recombinant proteins, a total of six immunizations. For primary immunization, 100 μl of 1 × 10 ⁶ NIH 3T3 IGF-IR cells were mixed with 100 μl Freund's Complete Immune Response Agent (CFA; Difco Laboratories, Detroit, USA). For all other immunizations, 100 μl of cells in PBS were used, or recombinant proteins were mixed with 100 μl Freund's Incomplete Immune Response Agent (ICFA; Difco).

Antigen specific ELISA

Anti-IGF-IR titer concentrations in the serum of immunized mice were measured by antigen specific ELISA. IGF-IR soluble extracellular domains were coated in 96 well plates overnight at 4 ° C., or at 37 ° C. for 2 hours at a concentration of 1 μg / ml in PBS. Thereafter, the wells were blocked with PBSTC (PBS supplemented with 0.05% Tween ^®- 20 and 2% chicken serum (Gibco BRL)) for 1 hour at room temperature. The first tap serum was diluted 1/50 in PBSTC and the serum from all other taps was prediluted to 1/100 in PBSTC and step diluted to 1/6400 or less. Diluted serum was added to the wells and incubated at 37 ° C. for 1 hour. Pre-tap serum was used as negative control. 200 ng / ml goat anti-human IGF-IR (100 μg / ml) was used as a positive control. The plates were then washed twice with PBST and with horse radish peroxidase (HRP) -conjugated rat anti-human IgG F (ab ') ₂ (DAKO) diluted 1/2000 in PBSTC. , Incubated at 37 ° C. for 1 hour. The wells were washed twice with PBST and 30 minutes in the dark with freshly prepared ABTS ^® solution (1 mg / ml) (ABTS: 2,2'-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) (RT) assay was developed at room temperature while absorbance was measured at 405 nm.

FACS  analysis

In addition to the measurement by antigen specific ELISA, anti-IGF-IR titer concentrations in the serum of immunized mice were also determined by FACS analysis. NIH 3T3 IGF-IR cells and untransfected NIH 3T3 IGF-IR cells were incubated with diluted serum at 4 ° C. for 30 minutes. Pre-tap serum was used as negative control. Initially, 200 ng / ml goat anti-human IGF-IR was used as a positive control. Cells were washed three times in PBS supplemented with 1% bovine serum albumin and 0.01% azide. The cells were then 4 ° C. with fluorescein isothiocyanate (FITC) -conjugated antigen binding fragment (F (ab ′) ₂ fragment) of rat anti-human human IgG diluted 1/100 in FACS buffer. Incubate for 30 minutes at. Cells were washed twice in FACS buffer and samples analyzed on FACSCalibur (Becton Dickinson, Erembodegem-Aalst, Belgium).

Mouse enhancer

If a serum titer of anti-IGF-IR is found to be sufficient, intravenously (iv) enhanced twice intravenously with 15 μg of IGF-IR extracellular domain in 200 μl PBS 4 and 3 days prior to additional fusion of mice. I was.

Hybridoma  Occur

Mice were sacrificed and spleen and lymph nodes located flanking the abdominal aorta and vena cava were collected. Fusion of spleen cells with lymph node cells and fusion partner SP 2.0 cells was performed according to standard procedures.

κ- ELISA

To determine if hybridomas generated from fusions give rise to human antibodies, κ-ELISA was performed. ELISA plates were coated by incubating at 4 ° C. overnight with rat anti-human IgG κ-light chain antibody (DAKO) diluted 1/10000 in PBS. After discarding the contents of the wells, the plates were blocked by incubating with PBSTC for 1 hour at room temperature. Thereafter, the wells were incubated with hybridoma culture supernatants diluted 1/2 in PBSTC. Culture medium diluted 1/2 in PBSTC was used as a negative control, and κ- light chain positive mouse serum diluted 1/100 in PBSTC was used as a positive control. The wells were then washed three times and incubated for 1 hour at 37 ° C. with HRP-conjugated rat anti-human IgG F (ab ′) ₂ (DAKO) diluted 1/2000 in PBSTC. The wells were washed three times and developed (RT) assay at room temperature for 30 minutes in the dark with freshly prepared ABTS ^® solution (1 mg / ml). Absorbance was measured at 405 nm in an ELISA plate reader.

Monoclonal antibodies 18 and 22 were prepared.

Example  2

IGF - IR  Anti- against IGF - IR  Measurement of the affinity of the antibody

Organization: BIACORE ^® 3000

Chip: CM5

Coupling: Amine Coupling

Buffer: HBS (HEPES, NaCI), pH 7.4, 25 ° C

For affinity measurements, anti human FCγ antibodies (from rabbits) were coupled to the chip surface for presentation of antibodies to IGF-IR. IGF-IR extracellular domains were added at various concentrations in solution. Association was measured by 3 minutes of IGF-IR-injection; Dissociation was measured by washing the chip surface with buffer for 5 minutes. Affinity data for antibodies 18 and 22 are shown in Table 1.

Table 1:

Affinity data measured by SPR (BIACORE ^® 3000)

Antibodies ka (1 / Ms) kd (1 / s) KD (M) 18 1.49 x 10 ⁵ 1.03 x 10 ^-7 6.95 x 10 ^-13 22 1.47 x 10 ⁵ 9.64 x 10 ^-5 6.56 x 10 ^-10

Example  3

Cell-cell-contact (3D culture) in  Three-dimensional growth of tumor cells and IGF Overexpression of -I Receptors

Material and method:

To study the effect on IGF-IR surface expression, NCI H322M cells were cultured in RPMI medium at low density or overfusion on optical grade glass cover slides. At the same time, H322M xenograft tissues isolated from control (untreated mice) were frozen in isopentane to impact and the cryosection cut to 5 μm thick. The immunofluorescence labeled mouse-anti IGF-IR monoclonal antibody (αIR3, 5 ㎍ / ml) or using the antibody according to the invention, followed by Cy3 (Amersham Biosciences, GB) or Alexa Fluor ^® 488 (Molecular Probes, Inc , USA), goat anti-mouse-antibody or goat anti-mouse antibody. Samples were imaged on a Leica SP2 confocal microscope or analyzed by FACS.

result:

When imaging H322M cells cultured at high density by confocal microscopy, it became apparent that IGF-IR clustered, particularly at the site of cell-cell contact. Compared to H322M cells grown in vivo, ie, in xenograft tissue, there was a significant similarity to the dense and in vitro culture as far as it relates to the organs of the surface IGF-I receptor.

Upregulation of surface IGF-I receptors in overfusion cultures of H322M cells was also quantified by FACS. When cells grew under high density conditions, IGF-I receptor surface expression increased by more than 10-fold compared to low density without significant cell-cell contact.

Other tumor cells, such as HT29, MDA231 and MCF-7, exhibited similar behaviors, indicating that upregulation of IGF-I receptors on the cell surface to make cell-cell contact sites is not a specific feature of H322M cells in vivo. It was shown to be a general characteristic of more tissues such as organs found (FIG. 1).

Example  4

Antibodies 18 r  In 3D cultures during processing IGF - IR  Expressive H322M  Suppression of growth of tumor cells ( WST - Assay )

H322M cells were cultured in RPMI1640 / 0.5% FCS medium on a poly-HEMA (poly (2-hydroxyethylmethacrylate)) coated dish to prevent adhesion to plastic surfaces. Under these conditions, H322M cells formed dense spheroids that grow in three dimensions (a property called anchorage independence). The spheroid closely resembles the three-dimensional histoarchitecture and organ of the solid tumor in situ. Spheroid cultures were incubated for 5 days in the presence of increasing amounts of antibody from 0-240 nM. Growth inhibition was measured using a WST conversion assay. When H322M spheroid cultures were treated with different concentrations of antibody 18 (1-240 nM), dose dependent growth inhibition could be observed (FIG. 2).

Example  5

In 3D culture IGF - IR  Expressing H322M  Suppression of growth of tumor cells ( Colony  Formation assay)

H322M cells were cultured in RPMI1640 / 10% NCS medium on poly-HEMA coated dishes to prevent adhesion to plastic surfaces. Under these conditions, H322M cells formed dense spheroids that grow in three dimensions (a property called fixed independence). The spheroids represent the three-dimensional histology and organs of the solid tumor in situ. Spheroid cultures were incubated for 5-10 days in the presence of increasing amounts of antibody at 0-7.5 μg / ml. <HBV> monoclonal antibody was used as a negative control. Colonies were imaged on a Zeiss Axiovert using phase difference and numbered using an automated imaging system (MetaMorph). When H322M spheroid cultures were treated with different concentrations (0.5-7.5 μg / ml) of antibody 18, dose dependent growth inhibition could be observed, whereas the control antibody <HBV> had little or no effect. Both the number and size of colonies were clearly reduced in culture treated with 7.5 μg / ml of antibody 18 (FIG. 3).

Quantitative analysis of colonies greater than 100 μm in diameter showed a decrease of about 66% in cultures treated with 0.5 μg / ml of antibody 18 (FIG. 4).

Example  6

IGF - IR  For tumor cells expressing IGF -I and IGF - II  Inhibition of binding

To determine the binding blocking ability of ligands IGF-I and IGF-II to the IGF-I receptor (IGF-IR) of the antibodies of the invention, competition experiments were performed with radiolabeled ligand peptides.

Human tumor cells (HT29, NCI H322M, 0.5-1 × 10 ⁵ / ml) were treated with 2 mM L-Glutamin, 1x non-essential amino acids (Gibco, Cat. No. 11140-035), 1 mM sodium pyruvate (Gibco, Cat. No. 11360-039) and 10% heat inactivated FCS (PAA, Cat. No. A15-771) were plated in RPMI 1640 medium (PAA, Cat. No. E15-039). For each experiment 20 ml cells in each medium were inoculated into six vessels of type T175 and incubated for 2 days at 37 ° C., 5% CO ₂ to obtain a fusion cell monolayer.

To collect each cell, 2 ml of 1 × Trypsin / EDTA (Gibco, Cat. No. 25300-054) per T175 flask was added and desorption of the cells was monitored by Zeiss Axiovert25 microscope. Cells were collected and medium of 10% FCS as described above was added to a total volume of 50 ml. Cells are trimmed by centrifugation at 1000 rpm (Heraeus sepatech, Omnifuge 2.0 RS) for 10 minutes and in 50 ml of binding buffer (120 mM NaCl, 5 mM KCl, 1.2 mM MgSO ₄ , 1 mM EDTA, 10 mM D ( +) Glucose, 15 mM NaAc, 100 mM Hepes pH 7.6, 1% BSA) resuspended. Cells were counted, cut off by centrifugation and adjusted to 1 × 10 ⁶ cells / ml with binding buffer.

I ¹²⁵ -labeled IGF-I and IGF-II peptides (Amersham, ˜2000 Ci / mmol, Cat. No. IM172 and IM238), dissolved in 0.1% CH ₃ COOH, 4 × 10 ⁵ counts / (min x ml) was diluted in binding buffer with final activity. 75 μl of antibody at specific concentrations were added to 200 μl of the cell suspension with 25 μl of prediluted I ¹²⁵ -labeled IGF-I or IGF-II peptide and incubated at 4 ° C. for 3.5 hours. Cells were trimmed by centrifugation at 2000 rpm (Eppendorf, 5415C) for 5 minutes and the supernatant removed. After washing twice in 1 ml binding buffer, cells were resuspended in 1 ml binding buffer and transferred to scintillation tubes. The amount of radioactive peptide bound to the cell surface receptor was measured with a scintillation counter.

The resulting IC ₅₀ curves are shown in FIGS. 5 and 6, indicating the ability of antibodies to inhibit the binding of IGF-I and IGF-II peptides to IGF-I receptors. The average IC ₅₀ value for antibody 18 is 0.3 nM. The results for the antibody αIR3 are shown in FIG. 7. No detectable inhibition of IGF-II binding could be observed.

Example  7

IGF - IR  Antibody Competition for Binding Assay

For epitope mapping of anti-IGF-IR monoclonal antibodies, a format similar to that for affinity measurement (Example 2) was chosen, but IGF-IR was pre-incubated for at least 0.5 hours with antibody in solution at room temperature (RT). I was. The mixture was injected and IGF-IR binding (or inhibition) was detected. The assay can measure the mutual inhibitory activity of monoclonal antibodies against IGF-IR binding. It was found that the antibodies of the present invention compete with the antibody, αIR3, which is known to bind aa 217-274, for binding to IGF-IR (Gustafson, TA, and Rutter, WJ, J. Biol. Chem. 265 ( 1990) 18663-18667).

Example  8

IGF -I Mediated IGF - IR  And Akt Of PKB  Inhibition of phosphorylation

To determine the ability of the antibodies of the present invention to inhibit the activation and phosphorylation of the IGF-I receptor (IGF-IR), a ligation experiment was performed with IGF-I peptides, followed by Western blots with antibodies specific for phosphorylated tyrosine. Lot analysis was performed.

Human tumor cells (HT29, NCI H322M, 5 x 10 ⁴ / ml), 2 mM L-Glutamin, 1x non-essential amino acids (Gibco, Cat. No. 11140-035), 1 mM sodium pyruvate (Gibco, Cat. No. 11360-039) and plated in RPMI 1640 medium (PAA, Cat. No. E15-039) supplemented with 0.5% heat inactivated FCS (PAA, Cat. No. A15-771). For the determination of IC ₅₀ values, 12 well plates for each experiment were seeded with 1 ml cells in separate media and incubated for 2 days at 37 ° C., 5% CO ₂ .

After 48 hours of incubation with low serum medium, the medium was carefully removed and replaced with different concentrations of diluted antibody in individual media. After 5 minutes incubation at 37 ° C., 5% CO ₂ , IGF-I peptide was added to a final concentration of 2 nM and cells were incubated again for 10 minutes under the conditions mentioned above. The medium was carefully removed by aspiration and 100 μl of cold lysis buffer per well was added (50 mM Hepes pH 7.2, 150 mM NaCl, 1 mM EGTA, 10% glycerol, 1% Triton ^® -X100, 100 mM NaF). , 10 mM Na ₄ P ₂ 0 ₇ , Complete ^® protease inhibitor). Cells were detached using a cell scraper (Corning, Cat. No. 3010) and the well contents transferred to an Eppendorf reaction tube. Cell fragments were removed by centrifugation at 4 ° C., 13000 rpm for 10 minutes, and half of the supernatant was added to 2 × Laemmli sample buffer in a 1: 1 (v / v) ratio. For immunoprecipitation of IGF-IR, 1 μl of polyclonal antibody against IGF-IRβ (C-20, Santa Cruz Biotechnologies) or the amino acid 440-586 of the extracellular domain (α-chain) of the human IGF Type 1 receptor Immediately before addition of the epitope-sensitive murine and monoclonal antibody (IgG1) (mAb 24-55, GroPep), the remaining supernatant of the cell lysate was spin-purged (4 min, 10 min at 13000 rpm). After incubation for 2 hours in a rotating Eppendorf reaction tube at 4 ° C., 25 μl Protein G Sepharose ^® beads (Amersham Biosciences, Cat. No. 17-0618-01) were added, followed by an additional 1 hour at 4 ° C. Was incubated. The antibody-protein-complexes with the beads and washed three times with and (4 ℃, 1 min at 2000 rpm), wash buffer (lysis buffer only 0.1% Triton ^® -X100) isolated by centrifugation. After boiling the beads in Laemmli sample buffer, the cell proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane (PROTRAN ^® BA 85, Schleicher & Schul) by semi-dry western blotting.

Phosphotyrosine specific antibodies (Upstate, clone 4G10, Cat. No. 05-321) were used to determine the phosphorylation status of immunopurified IGF-IR. For the detection of phosphorylated Akt / PKB, an antibody with specificity for phosphorylated Ser473 (Cell Signaling, Cat. No. 9271) was used.

The observation of the blocking of IGF-I induced phosphorylation of both IGF-IR and Akt / PKB is shown in FIG. 8.

Example  9

In vitro IGF - Of IR  Antibodies Mediated  Induction of Downregulation

To determine the effect of the antibodies of the present invention on the amount of IGF-I receptor (IGF-IR) in tumor cells, time course experiments and subsequent western blotting analyzes were performed with IGF-IR specific antibodies.

Human tumor cells (HT29, 5 × 10 ⁴ / ml), 2 mM L-Glutamin, 1x non-essential amino acids (Gibco, Cat. No. 11140-035), 1 mM sodium pyruvate (Gibco, Cat. No. 11360) -039) and RPMI 1640 medium (PAA, Cat. No. E15-039) supplemented with 10% heat inactivated FCS (PAA, Cat. No. A15-771). During each incubation period, 12 well plates for each experiment were incubated with 1 ml cells in separate media and incubated for 2 days at 37 ° C., 5% CO ₂ .

The medium was carefully removed and replaced with antibodies of different concentrations diluted in each medium. In two control wells, the medium was replaced with medium without antibody or medium with control antibody (AB-1, Oncogene, Cat. No. GR11). Cells were incubated at 37 ° C., 5% CO ₂ and individual plates were taken out for further processing after 15 minutes, 24 hours and 48 hours.

The medium was carefully removed by aspiration and 100 μl of cold lysis buffer added per well (50 mM Hepes pH 7.2, 150 mM NaCl, 1 mM EGTA, 10% glycerol, 1% Triton ^® -X100, 100 mM NaF). , 10 mM Na ₄ P ₂ 0 ₇ , Complete ^® protease inhibitor). Cells were detached using a cell scraper (Corning, Cat. No. 3010) and the well contents transferred to an Eppendorf reaction tube. Cell fragments were removed by centrifugation at 4 ° C., 13000 rpm for 10 minutes, and the supernatant was added to 2 × Laemmli sample buffer at a 1: 1 (v / v) ratio. Cellular proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (PROTRAN ^® BA 85, Schleicher & Schuell, Cat No. 10 401196) by semi-dry western blotting.

Protein concentration of IGF-IR was measured using an antibody specific for IGF-IR (C-20, Santa Cruz Biotechnologies, Cat. No. sc-713).

Downregulation of IGF-IR induced by the antibodies of the invention was observed less than 24 hours after antibody addition.

Example  10

3 expressing human insulin receptor T3 Inhibition of insulin binding to cells

In order to determine if the antibodies of the invention also block the binding of insulin to the insulin receptor (IR), competition experiments were conducted with radiolabeled ligand peptides.

Recombinantly 3T3 cells (1 × 10 ⁵ / ml) expressing a large number of (> 10 ⁵ ) human IR were transferred to 2 mM L-Glutamin (Gibco, Cat. No. 25030-024) and 10% heat inactivated FCS (PAA, Cat. No. A15-771) was plated in high concentration glucose (PAA, Cat. No. E15-009) MEM Dulbecco's medium (DMEM). For each experiment 20 ml cells in each medium were inoculated into six vessels of type T175 and incubated for 2 days at 37 ° C., 5% CO ₂ to obtain a fusion cell monolayer.

To collect individual cells, 2 ml of 1x Trypsin / EDTA (Gibco, Cat. No. 25300-054) per T175 flask was added and the detachment of the cells was monitored under a microscope. Cells were collected and medium of 10% FCS as described above was added to a total volume of 50 ml. Cells were trimmed by centrifugation at 1000 rpm for 10 minutes and in 50 ml of binding buffer (120 mM NaCl, 5 mM KCl, 1.2 mM MgSO ₄ , 1 mM EDTA, 10 mM D (+) glucose, 15 mM NaAc, 100 mM Hepes pH 7.6, 1% BSA) was resuspended. Cells were counted, cut off by centrifugation and adjusted to 1 × 10 ⁶ cells / ml with binding buffer.

I ¹²⁵ -labeled insulin peptide (Amersham, Cat. No. IM166, ˜2000 Ci / mmol), dissolved in 0.1% CH ₃ COOH, binding buffer with a final activity of 4 × 10 ⁵ counts / (min × ml) Diluted in water. 75 μl of antibody was added to 200 μl of the cell suspension with 25 μl of prediluted I ¹²⁵ -labeled insulin peptide (final antibody concentration 200 nM) and incubated at 4 ° C. for 3 or 5 hours. Cells were trimmed by centrifugation at 2000 rpm for 5 minutes and the supernatant removed. After washing twice in 1 ml binding buffer, cells were resuspended in 1 ml binding buffer and transferred to scintillation tubes. The amount of radioactive peptide bound to the cell surface receptor was measured with a scintillation counter.

The results show that the antibodies of the present invention do not interfere with the binding of insulin ligands to insulin receptors (FIG. 9).

Example  11

IGF - IR  And Akt Of PKB  Does not stimulate phosphorylation

To rule out the IGF-IR stimulatory activity of the antibodies of the invention, the phosphorylation of IGF-IR was measured in the absence of IGF-I ligands but in the presence of the antibodies and reference antibodies (αIR3, Oncogene, Germany) of the invention. This was done by Western-blotting analysis with phosphorylation-state specific antibodies. 3T3 cells transfected with IGF-IR (ATCC CRL 1658) (5 × 10 ⁴ cells / ml, Pietrzkowski, Z., et al., Cell Growth Differ. 4 (1992) 199-205) were subjected to 2 mM L-Glutamin (Gibco). , MEM Dulbecco medium (DMEM) of high glucose (PAA, Cat. No. E15-009) supplemented with 0.5% heat inactivated FCS (PAA, Cat. No. A15-771) Or human tumor cells (HT29, NCI H322M, 5 x 10 ⁴ / ml), 2 mM L-Glutamin, 1x non-essential amino acids (Gibco, Cat. No. 11140-035), 1 mM sodium pyruvate (Gibco, Cat No. 11360-039) and plated in RPMI 1640 medium (PAA, Cat. No. E15-039) supplemented with 0.5% heat inactivated FCS (PAA, Cat. No. A15-771). For determination of IC ₅₀ values, 1 ml cells in individual media were inoculated into 12 well plates for each experiment and incubated for 2 days at 37 ° C., 5% CO ₂ .

After 48 hours of incubation with low serum medium, the medium was carefully removed and replaced with different concentrations of antibody diluted in individual medium. Cells were incubated for 15 minutes under the conditions mentioned above. The medium was carefully removed by aspiration and 100 μl of cold lysis buffer added per well (50 mM Hepes pH 7.2, 150 mM NaCl, 1 mM EGTA, 10% glycerol, 1% Triton-X100, 100 mM NaF, 10 mM Na ₄ P ₂ 0 ₇ , Complete ™ Protease Inhibitor). Cells were detached using a cell scraper (Corning, Cat. No. 3010) and the well contents transferred to an Eppendorf reaction tube. Cell fragments were removed by centrifugation (Eppendorf centrifuge 5415R) at 4 ° C., 13000 rpm for 10 minutes, and half of the supernatant was added to 2 × Laemmli sample buffer in a 1: 1 (v / v) ratio. For immunoprecipitation of IGF-IR, just prior to addition of 1 μl of antibody to IGF-IR (C-20, Santa Cruz Biotechnologies, CatNo. Sc-713 or mAb 24-55, GroPep, CatNo. MAD1), The remaining supernatant of the cell lysate was subjected to clarification spin (10 min at 4 ° C, 13000 rpm). After 2 hours incubation at 4 ° C., in a rotating Eppendorf reaction tube, 25 μl Protein G Sepharose ™ beads (Amersham Biosciences, Cat. No. 17-0618-01) were added followed by an additional 1 hour at 4 ° C. Was incubated. The antibody-protein-complexes with the beads and washed three times with and (4 ℃, 1 min at 2000 rpm), wash buffer (lysis buffer only 0.1% Triton ^® -X100) isolated by centrifugation. After boiling the beads in Laemmli sample buffer, the cell proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (PROTRAN ^® BA 85, Schleicher & Schulel, Cat No. 10 401196) by semi-dry western blotting.

Phosphotyrosine specific antibodies (Upstate, clone 4G10, Cat No. 05-321, recognizing tyrosine-phosphorylated protein) were used to determine the phosphorylation status of immunopurified IGF-IR. For detection of phosphorylated Akt / PKB, an antibody against Akt1 with specificity for phosphorylated Ser473 (Cell Signaling, Cat. No. 9271) was used.

Akt / PKB kinase downstream in the signal pathway of IGF-IR was apparently activated by the reference antibody at concentrations above 5 nM, but was not activated by the antibodies of the invention at concentrations below 10,000 nM. The results are shown in FIGS. 10 and 11 (low concentration serum medium of HM = 0.5% FCS, low concentration serum medium of HM + IGFI = 0.5% FCS and 10 nM hIGF-I).

Example  12

H322M  Induction of Receptor Down-regulation in Xenograft Models

Tumors were induced in nude mice and treated with different concentrations of the antibody of the invention once. Twenty four hours after treatment, tumors were extracted and homogenized under liquid nitrogen. Cool Lysis Buffer was added at a 3: 1 buffer-volume to tumor-weight ratio (50 mM Hepes pH 7.2, 150 mM NaCl, 1 mM EGTA, 10% Glycerol, 1% Triton-X100, 100 mM NaF, 1 mM) Na ₃ V0 ₄ , 10 mM Na ₄ P ₂ 0 ₇ , Complete ™ protease inhibitor, 1 mM PMSF), thawing tumor homogenate and complete mixing. After dissolving the tissue for 15 minutes on ice, insoluble fragments were removed by centrifugation (Eppendorf centrifuge 5415R) at 4 ° C., 13000 rpm for 10 minutes. Protein concentration of the samples was measured by Micro BCA ™ Reagents (Pierce) and adjusted to the same concentration by addition of lysis buffer. A portion of the supernatant was added to 2 × Laemmli sample buffer at a 1: 1 (v / v) ratio. Cellular proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (PROTRAN BA 85, Schleicher & Schuell, Cat No. 10 401196) by semi-dry western blotting. IGF-IR was detected using an IGF-IR specific antibody (C-20, Santa Cruz Biotechnologies, Cat No. sc-713).

When treated with the antibodies of the invention, a decrease in the dependence of IGF-IR concentrations was observed with EC50 evaluated at 0.6 mg / kg (FIG. 12).

Example  13

C1q  Combination ELISA

Introduction

To determine the ability of the antibody according to the invention to fix C1q, an ELISA approach was used. C1q is part of the adaptive immune system and binds to the immune complex, causing a series of activation of several zymogens. Enzymes, in turn, cause cleavage of C3 molecules that can initiate inflammatory responses, opsonization of foreign or abnormal particles, and lysis of cell membranes.

In principle, human C1q is added to ELISA plates coated in the concentration range of the antibody. C1q binding is detected by antibody to human C1q and then by peroxidase-labeled conjugate.

Materials and methods

Antibodies 18, 8 and 23 and control antibodies were tested at concentrations of 10, 5, 1 and 0.5 μg / ml. Table 1 shows the specificity of the samples tested. As a negative control, human IgG4 (CLB, stock 0.5 μg / ml) that binds very weakly to C1q was used. Human IgG1 was incorporated as a positive control. Human C1q stock solution at a concentration of 2 μg / ml was used. For detection of C1q, anti-rabbit IgG antibodies conjugated with rabbit antibody (Dako) and horseradish peroxidase (Sigma) against C1q were used.

Calculations and Curves fitting

Calculations related to the maximum binding (Bmax) of HuMAbs tested were measured using nonlinear regression curve fitting (one site binding) using Graphpad Prism software.

result

Antibodies according to the invention exhibit dose dependent binding of human C1q protein. The optical density at 405 nm (OD 405 nm) was plotted against HuMAb concentration and the curves were fitted using nonlinear regression. The most suitable values for maximum binding (Bmax), along with the correlation coefficient (R2) and standard deviation of the curve for each value, are listed in Table 2. The lowest correlation coefficient had a value of 0.950 (IgG4). The maximum binding is 0.279, human IgG4 (negative control) shows the minimum binding of C1q. As shown the maximum binding of 1.729 and 2.223, respectively, both positive controls IgG1 and IgG3 bound to C1q.

TABLE 2

C1q  Combination ELISA  (n = 3) in Tested Of HuMAb  Max Combined ( Bmax )

Most suitable value Bmax Standard deviation Bmax Goodness of fit R ² Standard deviation R ² IgG1 1.729 0.166 0.983 0.010 IgG3 2.223 0.947 0.995 0.005 IgG4 0.279 0.280 0.950 0.041 Antibody 18 1.670 0.601 0.988 0.005

Correlation coefficients (R2) and standard deviations are also listed.

Compared to the C1q binding of human IgG4 (negative control with O.D. 0.279), all antibodies tested can equally immobilize C1q.

Example  14

term- IGF - IR HuMAb  Antibodies by Mediated  Measurement of effector function

To determine the ability to induce the immune effector mechanism of the generated HuMAb antibody, complement dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) studies were performed.

To study the CDC (National Cancer Institute, lung adenocarcinoma cell line), H322M, H460 and NIH 3T3 cells (2-6 × 10 ⁶ ) were treated with 100 μCi ⁵¹ Cr (Amersham Pharmacia Biotech, UK, Cat CJS11) for 45-120 minutes. Was labeled. After labeling, cells were washed twice with 40 ml PBS and spun for 3 minutes at 1500 rpm. Cells were then plated at 5,000 per well in round bottom plates at a volume of 50 μl. Antibodies were added to 50 μl cell suspension at a final concentration ranging from 25-0.1 μg / ml in the volume of 50 μl cell culture medium and incubated for 30-60 minutes. After incubation, excess antibody was removed by washing twice with PBS. 100 μl of active or inactive (30 min at 56 ° C.) congested human serum, guinea pig, rabbit or nude mouse serum diluted to 1/3 to 1/30 was added and the cells were incubated for 3 hours and then the cells Was spun at 1500 rpm for 3 minutes. 100 μl of the supernatant was collected, transferred to a polypropylene tube, and the value was measured by γ-counter.

To determine the effect of the antibody on ADCC, H322M, H460 and NIH 3T3 or other suitable IGF-IR expressing cells (2-6 x 10 ⁶ ) were labeled with 100 μCi ⁵¹ Cr for 45-120 minutes (Amersham Pharmacia Biotech, UK, Cat CJS11), washed twice with 40 ml of PBS and spun for 3 minutes at 1500 rpm. Cells were plated at a volume of 50 μl, 5,000 per well in round bottom plates. HuMAb antibody was added to 50 μl cell suspension at a final concentration ranging from 25-0.1 μg / ml in the volume of 50 μl cell culture medium and incubated for 15 minutes. 50 μl of effector cells, PBMCs or purified effector cells, freshly isolated from the smokescreen, were then added with an E: T ratio in the range of 100: 1 to 5: 1. Plates were centrifuged for 2 minutes at 500-700 rpm and incubated overnight at 37 ° C. After incubation, cells were spun down at 1500 rpm for 3 minutes, 100 μl of supernatant was collected, transferred to a polypropylene tube and measured with a γ-counter.

The extent of cell lysate by CDC or ADCC is expressed as the highest percentage of radioactive release from target cells lysed by detergent, corrected for spontaneous release of radioactive from each target cell.

Example  15

H322M  Tumor growth inhibition

The effect of antibody 18 in vivo was studied by inducing tumors in nude mice without thymus following established methods. Human H322M NSCLC cells were co-injected subcutaneously with Matrigel into nude mice (nu / nu) of 6-7 week-old thymus. For this purpose, 5 × 10 ⁶ H322M cells were concentrated in 100 μl of culture medium and mixed with 100 μl Matrigel. 200 μl of this mixture was injected into the right flank of mice. Tumor volume was calculated by Geran et al. ("Protocols for screening chemical agents and natural products against animal tumors and other biological systems", Cancer Chemother. Rep. 11.301, 1972), tumor volume [mg] = (length x According to (width) ² ), it was calculated by measuring the tumor diameter twice a week with a Vernier caliper. Antibody was administered intraperitoneally (ip) at 10 ml / kg. Treatment was initiated by administering a double dose of antibody to a doubled volume. Tumors were induced in nude mice as described above. After tumors have grown to an average volume of 160 mg, mice are given 6, 0.6 and 0.06 mg /, six times a week, in successive doses starting with 12, 1.2 and 0.12 mg / kg with the loading dose given once on Day 1 of treatment. kg of antibody were treated intraperitoneally. FIG. 13 is a graphical representation of tumor volume measured during treatment up to day 67 where the animals were sacrificed and the experiment ended. Experiments show that blocking of the IGF-IR axis by rhu anti-IGF-IR mAb 18 results in antitumor effects when administered alone at 6 and 0.6 mg / kg. In contrast, 0.06 mg / kg had no effect on tumor growth.

In addition, antibody 18 was tested in combination with gemcitabine in the same model. Tumors were induced as described above, and in all groups tumors were built up to an average of 170 mm ³ and treatment started when grown. Antibodies were administered ip once a week in combination with 6 and 0.6 mg / kg, and 0.6 mg to 62 mg / kg of gemcitabine. Gemcitabine was administered four times in one cycle, once every three days. Again, treatment was started by administering a doubled dose of antibody. 14 shows tumor size associated with various treatments over time. Experiments have shown that the treatment of antibody 18 administered once every seven days inhibits tumor growth on its own and increases the effect of gemcitabine, a known anti-metabolic compound.

Example  16

3 T3  Tumor growth inhibition

Tumors were induced in nude mice essentially as described in Example 15, except for using murine and 3T3 fibroblasts expressing human IGF-IR. Mice constructing approximately 180 mg of tumor were treated intraperitoneally with 18, 6 or 0.6 mg / kg of antibody 18 once a week. Again, treatment was started with doubled doses of antibody given at loading doses (36, 12 and 1.2 mg / kg). Experiments show that treatment of antibodies can delay tumor growth when administered at 18 and 6 mg / kg once a week (FIG. 15).

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WO 02/053596

WO 87/05330

WO 92/03918

WO 92/22645

WO 93/1227

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Claims (19)

As an antibody that binds to IGF-IR and inhibits the binding of IGF-I and IGF-II to IGF-IR, the following a), b), c), d): a) of IgG1 isotype, b) the ratio of IC ₅₀ values of inhibition of binding of IGF-I to IGF-IR to inhibition of binding of IGF-II to IGF-IR is shown as 1: 3 to 3: 1, c) In a cell phosphorylation assay using HT29 cells in medium containing 0.5% heat inactivated fetal bovine serum (FCS), compared to this assay performed without the antibody, IGF- at a concentration of 5 nM Inhibits IR phosphorylation by more than 80%, d) in a cell phosphorylation assay using 3T3 cells that provide 400,000 to 600,000 molecules of IGF-IR per cell in medium containing 0.5% heat inactivated fetal bovine serum (FCS) compared to this assay performed without the antibody. If present, does not exhibit IGF-IR stimulatory activity measured by IGF-IR phosphorylation at a concentration of 10 μM; An antibody comprising the following sequences: i) an antibody heavy chain comprising SEQ ID NO: 1 or 3; ii) an antibody light chain comprising SEQ ID NO: 2 or 4. The antibody of claim 1, wherein said antibody induces apoptosis of at least 20% of the cells of an IGF-IR expressing cell sample after 24 hours at a concentration of 100 nM by said ADCC. The antibody of claim 1 or 2, wherein said antibody induces at least 20% cell death of an IGF-IR expressing cell sample after 4 hours at a antibody concentration of 100 nM of said antibody by CDC. The antibody according to claim 1 or 2, which is a human or humanized antibody. The antibody of claim 1 or 2, characterized by an affinity of about 10 ⁻¹³ to 10 ⁻⁹ M (K _D ). delete The antibody according to claim 1 or 2, which can be obtained from the hybridoma cell line <IGF-1R> HUMAB Clone 18 (DSM ACC 2587) or <IGF-1R> HUMAB Clone 22 (DSM ACC 2594). delete A pharmaceutical composition for antitumor treatment containing the antibody according to claim 1 or 2 in a pharmaceutically effective amount. Hybridoma cell lines <IGF-1R> HUMAB Clone 18 (DSM ACC 2587) and <IGF-1R> HUMAB Clone 22 (DSM ACC 2594). A method for preparing a pharmaceutical composition for treating antitumor, comprising a pharmaceutically effective amount of the antibody according to claim 1. Nucleic acid encoding a polypeptide that can be assembled with each other antibody chain as defined in a) or b) below, wherein the polypeptide is either a) or b): a) an antibody heavy chain comprising SEQ ID NO: 1 or 3; b) an antibody light chain comprising SEQ ID NO: 2 or 4. An expression vector comprising said nucleic acid, capable of expressing the nucleic acid according to claim 12 in a prokaryotic or eukaryotic host cell. Prokaryotic or eukaryotic host cell comprising the vector of claim 13. A method for producing a polypeptide that binds to IGF-IR and inhibits the binding of IGF-I and IGF-II to IGF-IR, comprising encoding a nucleic acid and a light chain encoding the heavy chain according to claim 12 in prokaryotic or eukaryotic host cells. Expressing a nucleic acid to recover the polypeptide from the cell. delete delete 3T3 cells providing 400,000 to 600,000 molecules of IGF-IR per cell in medium containing 0.5% heat inactivated fetal bovine serum (FCS) as a method of screening one antibody against IGF-IR from the majority of antibodies to IGF-IR. Cell phosphorylation assays using a majority of the antibodies and when compared to these assays performed without the antibody, select antibodies that do not exhibit IGF-IR stimulatory activity as measured by PKB phosphorylation at a concentration of 10 μM. Characterized in that the method. delete

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